Stilbenoids and Flavonoids from Cajanus cajan (L.) Millsp. and Their α-Glucosidase Inhibitory Activities

Two new stilbenoids, cajanstilbenoid C (1) and cajanstilbenoid D (2), together with eight other known stilbenoids (3-10) and seventeen known flavonoids (11-27), were isolated from the petroleum ether and ethyl acetate portions of the 95% ethanol extract of leaves of Cajanus cajan (L.) Millsp. The planar structures of the new compounds were elucidated by NMR and high-resolution mass spectrometry, and their absolute configurations were determined by comparison of their experimental and calculated electronic circular dichroism (ECD) values. All the compounds were assayed for their inhibitory activities against yeast α-glucosidase. The results demonstrated that compounds 3, 8-9, 11, 13, 19-21, and 24-26 had strong inhibitory activities against α-glucosidase, with compound 11 (IC50 = 0.87 ± 0.05 μM) exhibiting the strongest activity. The structure–activity relationships were preliminarily summarized. Moreover, enzyme kinetics showed that compound 8 was a noncompetitive inhibitor, compounds 11, 24-26 were anticompetitive, and compounds 9 and 13 were mixed-competitive.


Introduction
Diabetes mellitus (DM) is a metabolic disorder induced by multiple causes and characterized by persistent hyperglycemia. With improved living standards and altered lifestyles, the number of diabetes patients is on the rise and is expected to reach 300 million by 2025, which will rank diabetes as the third most common disease that seriously endangers human health and causes a huge economic burden, right after cardiovascular diseases and malignant cancer [1]. Diabetes is categorized into types I and II, and the number of patients suffering from type II accounts for more than 90% [2]. The available oral drugs in clinical use for the treatment of diabetes include biguanides, sulfonylureas, insulin sensitizers, glinide insulin secretagogue, α-glucosidase inhibitors, dipeptidyl peptidase-IV (DPP-4) inhibitors, and sodium-glucose cotransporter 2 (SGLT-2) inhibitors [3]. α-Glucosidase inhibitors exert their hypoglycemic effect by competitively inhibiting α-glucosidase located in the small intestine to slow down the decomposition of starch into glucose. At present, α-glucosidase inhibitors mainly include acarbose, voglibose, and miglitol [4]. However, the preparation process of those oral hypoglycemic agents is complicated and the cost for long-term use is high, and their long-term use often causes abdominal discomforts [5]. Therefore, it is urgent to find new α-glucosidase inhibitors that might eliminate these problems.
Cajanus cajan (L.) Millsp, an erect shrub or subshrub, also known as pigeon pea, is the sixth-largest edible bean in the world. C. cajan is mainly distributed in South and Southeast

Results and Discussion
Based on our previous results from the same plant collected from Guizhou [10], flavonoids might be promising α-glucosidase inhibitors, and they are mainly present in petroleum ether and ethyl acetate parts extracted from 95% ethanol crude extract; thus, herein, the dried leaves of C. cajan (30 kg) were extracted with 95% ethanol. The extraction solvent was then evaporated with a rotatory evaporator to give a crude extract; the crude extract was dissolved in water and successively partitioned with petroleum ether and

Results and Discussion
Based on our previous results from the same plant collected from Guizhou [10], flavonoids might be promising α-glucosidase inhibitors, and they are mainly present in petroleum ether and ethyl acetate parts extracted from 95% ethanol crude extract; thus, herein, the dried leaves of C. cajan (30 kg) were extracted with 95% ethanol. The extraction solvent was then evaporated with a rotatory evaporator to give a crude extract; the crude extract was dissolved in water and successively partitioned with petroleum ether and ethyl acetate to afford petroleum ether part (835.6 g) and ethyl acetate part (456.4 g). Petroleum ether and ethyl acetate extracts being subjected to purification processes yielded ten stilbenoids (1-10) and seventeen flavonoids (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27). The structures of new compounds (1)(2) were characterized by HR-MS,1D-NMR, 2D-NMR, and ECD, while the structures of known compounds  were determined mainly through comparison of 1D-NMR data with those reported in the literature. In order to obtain promising α-glucosidase inhibitors, all the purified compounds were screened for their α-glucosidase inhibitory activity, and the ones that showed robust activity were further elucidated for their inhibition type by Lineweaver-Burk plots.

Structural Elucidation
Compound 1 was a yellow powder. From its quasi-molecular ion peak at m/z 377.1354 (calcd. for m/z 377.1359, [C 21 H 22 O 5 Na] + ) on positive ion HR-ESI-MS ( Figure S1), its molecular formula was indicated as C 21 H 22 O 5 ; thus, an unsaturation degree of 11 was calculated. By comparing both its 1 H-NMR and 13 C-NMR data (Table 1 and Figures S4 and S5) with those of other known stilbenes, we isolated (3-10); we suspected it was also a derivative of stilbene. In the 1 H-NMR data, the signal at δ H 6.45 (1H, s) and peaks at δ H 7.43 (2H, d, J = 6.9 H Z ), 7.34 (2H, t, J = 7.6 Hz), and 7.24 (1H, t, J = 7.4 Hz) suggested the existence of two benzene rings.  [11,12], suggesting that the skeleton of 1 was the same as Carexane O, which was also supposed to be biogenetically derived from stilbene. The differences between 1 and Carexane O were that instead of the presence of two hydroxy groups on the benzene ring, one of the hydroxyls changed into a methoxy group, and there was also an additional carbonyl group. Moreover, the unsaturation number of 1 is 11, and Carexane O is 10, which implies that instead of a free hydroxy group on the C2 between the two benzene rings, it might be cyclized into an ester ring with its adjacent hydroxy group, which was confirmed by the molecular weight of 354 (deduced from its quasi-molecular ion at 377.1354 [M+Na] + ). To further evidence this cyclization, HMBCs of δ H 5.22/δ C 162.1, 145.5 were found, and other key HMBCs are also shown in Figure 2. Thus, the planar structure of compound 1 was established. The configuration of H-7 and H-8 was deduced to be trans according to their large H-H coupling constant (J = 10.5 Hz), while the configuration of H-8 and H-2" was elucidated to be cis by the correlation [2.30 (dd, J = 10.5, 7.7 Hz, H-8)/2.46 (t, J = 8.6 Hz, H-2")] found on the NOESY spectrum ( Figure S8). The experimental ECD spectrum of compound 1 agreed with its calculated data of the (7R, 8R, 2"S) configuration ( Figure 3a); therefore, the absolute configuration of compound 1 (structure shown in Figure 1) was elucidated as (7R, 8R, 2"S). Thus, compound 1 was determined to be new, and given the name of cajanstilbenoid C.
Compound 2 was yellow amorphous powder; from its quasi-molecular ion peak at m/z 377.1354 (calcd. for m/z 377.1359, [C 21 H 22 O 5 Na] + ) on positive HR-ESI-MS ( Figure S9), its molecular formula was indicated as C 21 H 22 O 5 , and an unsaturation degree of 11 was calculated. In the 1 H-NMR data ( Table 1 and Figure S12 (Table 1), suggested the presence of a hydroxylated isopentyl moiety, which was cyclized into a six-membered ring with its ortho hydroxy group. The NMR data of 2 were found to resemble those of Chiricaine B [13], except that for 2, a hydroxy group changed into a methoxy group, and there was an additional signal of a carbonyl. The location of the methoxy group was determined by the following HMBC correlations: δ H 3.88/δ C 157.9; δ H 2.41 (1H, dd, J = 17.4, 7.6 Hz), 2.79 Molecules 2023, 28, 3779 4 of 11 (1H, dd, J = 17.4, 5.5 Hz)/δ C 157.9. The location of the carbonyl was settled by the HMBC correlation of δ H 6.90 (1H, s) with δ C 109.4 (key HMBC correlations are shown in Figure 2). The experimental ECD spectrum of compound 2 agreed with its calculated data of 2"R configuration (Figure 3b), so the absolute configuration of compound 2 (structure shown in Figure 1) was elucidated as 2"R. Thus, compound 2 was determined to be new, and given the name cajanstilbenoid D. with those reported in the literature. In order to obtain promising α-glucosidase inhibitors, all the purified compounds were screened for their α-glucosidase inhibitory activity, and the ones that showed robust activity were further elucidated for their inhibition type by Lineweaver-Burk plots.

Structural Elucidation
Compound 1 was a yellow powder. From its quasi-molecular ion peak at m/z 377.1354 (calcd. for m/z 377.1359, [C21H22O5Na] + ) on positive ion HR-ESI-MS ( Figure S1), its molecular formula was indicated as C21H22O5; thus, an unsaturation degree of 11 was calculated. By comparing both its 1 H-NMR and 13 C-NMR data (Table 1 and Figures S4 and  S5) with those of other known stilbenes, we isolated (3-10); we suspected it was also a derivative of stilbene. In the 1 H-NMR data, the signal at δH 6.45 (1H, s) and peaks at δH 7.43 (2H, d, J = 6.9 HZ), 7.34 (2H, t, J = 7.6 Hz), and 7.24 (1H, t, J = 7.4 Hz) suggested the existence of two benzene rings. Two methyl groups were indicated according to the peaks of δH 0.68 (s, 3H), 0.66 (s, 3H) on 1  In addition, the NMR data were in great similarity with those of Carexane O [11,12], suggesting that the skeleton of 1 was the same as Carexane O, which was also supposed to be biogenetically derived from stilbene. The differences between 1 and Carexane O were that instead of the presence of two hydroxy groups on the benzene ring, one of the hydroxyls changed into a methoxy group, and there was also an additional carbonyl group. Moreover, the unsaturation number of 1 is 11, and Carexane O is 10, which implies that instead of a free hydroxy group on the C2 between the two benzene rings, it might be cyclized into an ester ring with its adjacent hydroxy group, which was confirmed by the molecular weight of 354 (deduced from its quasi-molecular ion at 377.1354 [M+Na] + ). To further evidence this cyclization, HMBCs of δH 5.22/ δC 162.1, 145.5 were found, and other key HMBCs are also shown in Figure 2. Thus, the planar structure of compound 1 was established. The configuration of H-7 and H-8 was deduced to be trans according to their large H-H coupling constant (J = 10.5 Hz), while the configuration of H-8 and H-2" was elucidated to be cis by the correlation [2.30 (dd, J = 10.5, 7.7 Hz, H-8)/2.46 (t, J = 8.6 Hz, H-2")] found on the NOESY spectrum ( Figure S8). The experimental ECD spectrum of compound 1 agreed with its calculated data of the (7R, 8R, 2"S) configuration ( Figure 3a); therefore, the absolute configuration of compound 1 (structure shown in Figure 1) was elucidated as (7R, 8R, 2"S). Thus, compound 1 was determined to be new, and given the name of cajanstilbenoid C.  In order to obtain promising α-glucosidase inhibitors, all the purified compounds were screened for their α-glucosidase inhibitory activity, and the ones that showed robust activity were further elucidated for their inhibition type by Lineweaver-Burk plots.

Structural Elucidation
Compound 1 was a yellow powder. From its quasi-molecular ion peak at m/z 377.1354 (calcd. for m/z 377.1359, [C21H22O5Na] + ) on positive ion HR-ESI-MS ( Figure S1), its molecular formula was indicated as C21H22O5; thus, an unsaturation degree of 11 was calculated. By comparing both its 1 H-NMR and 13 C-NMR data (Table 1 and Figures S4 and  S5) with those of other known stilbenes, we isolated (3-10); we suspected it was also a derivative of stilbene. In the 1 H-NMR data, the signal at δH 6.45 (1H, s) and peaks at δH 7.43 (2H, d, J = 6.9 HZ), 7.34 (2H, t, J = 7.6 Hz), and 7.24 (1H, t, J = 7.4 Hz) suggested the existence of two benzene rings. Two methyl groups were indicated according to the peaks of δH 0.68 (s, 3H), 0.66 (s, 3H) on 1  In addition, the NMR data were in great similarity with those of Carexane O [11,12], suggesting that the skeleton of 1 was the same as Carexane O, which was also supposed to be biogenetically derived from stilbene. The differences between 1 and Carexane O were that instead of the presence of two hydroxy groups on the benzene ring, one of the hydroxyls changed into a methoxy group, and there was also an additional carbonyl group. Moreover, the unsaturation number of 1 is 11, and Carexane O is 10, which implies that instead of a free hydroxy group on the C2 between the two benzene rings, it might be cyclized into an ester ring with its adjacent hydroxy group, which was confirmed by the molecular weight of 354 (deduced from its quasi-molecular ion at 377.  Figure S8). The experimental ECD spectrum of compound 1 agreed with its calculated data of the (7R, 8R, 2"S) configuration ( Figure 3a); therefore, the absolute configuration of compound 1 (structure shown in Figure 1) was elucidated as (7R, 8R, 2"S). Thus, compound 1 was determined to be new, and given the name of cajanstilbenoid C. In order to obtain promising α-glucosidase inhibitors, all the purified compounds were screened for their α-glucosidase inhibitory activity, and the ones that showed robust activity were further elucidated for their inhibition type by Lineweaver-Burk plots.

Inhibitory Kinetics of Compounds 8-9, 11, 13, 24-26 against α-Glucosidase
The inhibitory kinetics of seven compounds (8-9, 11, 13, 24-26) that have strong αglucosidase inhibitory activities were studied. The Lineweaver-Burk plot was used to determine their inhibition types against α-glucosidase. As shown in Figure 4, all the lines of compounds 11, 24, 25, and 26 are almost parallel. As the concentrations of compounds 11, 24, 25, and 26 decreased, V max (maximum reaction rate) and K m (Michaelis constant) decreased, but K m /V max remained unchanged; in this case, the way compounds 11, 24, 25, and 26 mediated α-glucosidase inhibition was anticompetitive, which means that the compounds do not directly bind to the free enzyme, but only bind to the enzyme-substrate complex, thereby interrupting the enzymatic reaction. As for compound 8, the slope of the lines became greater with the increase in the concentration, but all straight lines intersected with the x-axis almost at one point (while V max decreased, K m was unchanged), showing that α-glucosidase inhibition mediated by compound 8 is noncompetitive, signifying that it can bind to either the free α-glucosidase or the enzyme-substrate complex to interfere the enzyme reaction without directly blocking the binding of the substrate to the enzyme. For compounds 9 and 13, the slope of the lines also became greater when their concentrations increased, but all the lines intersected in the third quadrant (K m decreased with V max) , indicating that compounds 9 and 13 mediated α-glucosidase inhibition in a mixed way, in which case, inhibitors can bind to either the enzyme or enzyme-substrate complex.
showing that α-glucosidase inhibition mediated by compound 8 is noncompetitive, signifying that it can bind to either the free α-glucosidase or the enzyme-substrate complex to interfere the enzyme reaction without directly blocking the binding of the substrate to the enzyme. For compounds 9 and 13, the slope of the lines also became greater when their concentrations increased, but all the lines intersected in the third quadrant (Km decreased with Vmax), indicating that compounds 9 and 13 mediated α-glucosidase inhibition in a mixed way, in which case, inhibitors can bind to either the enzyme or enzyme-substrate complex.
For stilbenoids, the activity of the ones that have hydroxy and methoxy substituents at C-3 and C-5 is relatively high, and the activity of the one possessing a methoxy group at C-5 is stronger than the one with a hydroxyl group at this position. In addition, the isopentenyl substituent favors activity, and when ring A contains a carboxyl or ester group, the activity decreases.
For flavonoids, their type (flavones or isoflavones), glycosylation, and substituents (positions of hydroxy, methoxy groups, and isopentenyl moiety) all affect their inhibitory
For stilbenoids, the activity of the ones that have hydroxy and methoxy substituents at C-3 and C-5 is relatively high, and the activity of the one possessing a methoxy group at C-5 is stronger than the one with a hydroxyl group at this position. In addition, the isopentenyl substituent favors activity, and when ring A contains a carboxyl or ester group, the activity decreases.
For flavonoids, their type (flavones or isoflavones), glycosylation, and substituents (positions of hydroxy, methoxy groups, and isopentenyl moiety) all affect their inhibitory effects against α-glucosidase. Basically, the activity of flavones was stronger than that of isoflavones, and when the hydroxyl groups were substituted at C-5 and C-7, and the isopentenyl group was substituted at C-8, the inhibition effect was enhanced, whereas the activity was reduced when the hydroxyl group was glycosylated. The activity of the B ring with the hydroxyl group on C-3 and C-4 was stronger than that of no substituent on the B ring.

General Experimental Procedures
NMR data were obtained by Bruker Avance Neo-400 MHz NMR spectrometer (Bruker, Germany); a VG-Autospec-3000 mass spectrometer (Beckman Coulter, Inc. America) was adopted to acquire HR-ESIMS spectra. A Fourier transform infrared spectrometer FTIR-650 from Tianjin Gangdong Technology Development Co., Ltd. (Tianjin, China) was employed to perform infrared spectra. UV spectra were documented by UV/Vis spectrophotometer UV-2700 (Shimadzu Instrument Suzhou Co., Ltd., Suzhou, China). TLC plates of Silica gel GF254 were purchased from Yantai Jiangyou Silicon Development Company (Yantai, China), and spots were observed by being exposed under UV light or heated after being sprayed with H 2 SO 4 dissolved in EtOH (5% v/v). Purification by HPLC was carried out with LC-20AR pumps and an SPD-M20A UV detector (Shimadzu, Kyoto, Japan). With a J-810 CD spectrometer from JASCO, Ltd. (Tokyo, Japan), ECD spectra were measured. The optical rotation was detected under the polarimeter-Autopol VI (Yunnan Gaosheng Import & Export Co., Ltd., Yunnan, China). A BioTek ELX800 microplate reader (USA) was used to measure absorbance.

Plant Material
The plant materials were purchased on 29 September 2020 from Taifu Agriculture and Forestry Technology Company (Honghe County, Yunnan Province, China), and identified by associate professor Shaohuan Liu to be leaves of C. cajan (L.) Millsp. A voucher specimen (20200929) was preserved at the Herbarium of School of Pharmaceutical Sciences, Guizhou Medical University.

Extraction and Isolation
The leaves of C. cajan (30 kg) were extracted by reflux with 95% ethanol 3 times, for 2 h each time, and the combined ethanol was concentrated to obtain crude extract. After being suspended with water, the crude extract was partitioned with petroleum ether and ethyl acetate successively to obtain petroleum ether part (835.6 g) and ethyl acetate part (456.4 g).

Assay for α-Glucosidase Inhibitory Activity of Compounds 1-27
The compounds 1-27 were evaluated for their inhibitory activities against α-glucosidase by employing the method of Lei [10]. This experiment was carried out in 96-well plates with reaction system of 230 µL. Firstly, 95 µL of PBS buffer solution (0.1 mol/L pH 6.8) was put into each well, then 5 µL of different concentrations (0.0625, 0.125, 0.25, 0.5, 1.0, 2.0 mmol/L) of acarbose solution or compounds 1-27 were added, then 30 µL of 0.6 U/mL α-glucosidase was pipetted and mixed by light shake. The 96-well plates were incubated in a constant-temperature incubator for 20 min at 37 °C, then 20 µL of 2.658 mmol/L p-PNG was added, and then incubation continued for another 28 min. The termination of the reaction was conducted by adding 80 µL of 0.2 mol/L Na2CO3 to each well. The absorbance was recorded for each well at 405 nm by a microplate reader, and the experiment was carried out three times in parallel. The measured absorbance values were processed by Graphpad Prism software 9.5 to calculate the IC50 values of the compounds. 8-9, 11, 13

Assay for α-Glucosidase Inhibitory Activity of Compounds 1-27
The compounds 1-27 were evaluated for their inhibitory activities against α-glucosidase by employing the method of Lei [10]. This experiment was carried out in 96-well plates with reaction system of 230 µL. Firstly, 95 µL of PBS buffer solution (0.1 mol/L pH 6.8) was put into each well, then 5 µL of different concentrations (0.0625, 0.125, 0.25, 0.5, 1.0, 2.0 mmol/L) of acarbose solution or compounds 1-27 were added, then 30 µL of 0.6 U/mL α-glucosidase was pipetted and mixed by light shake. The 96-well plates were incubated in a constant-temperature incubator for 20 min at 37 °C, then 20 µL of 2.658 mmol/L p-PNG was added, and then incubation continued for another 28

Assay for α-Glucosidase Inhibitory Activity of Compounds 1-27
The compounds 1-27 were evaluated for their inhibitory activities against α-glucosidase by employing the method of Lei [10]. This experiment was carried out in 96-well plates with reaction system of 230 µL. Firstly, 95 µL of PBS buffer solution (0.1 mol/L pH 6.8) was put into each well, then 5 µL of different concentrations (0.0625, 0.125, 0.25, 0.5, 1.0, 2.0 mmol/L) of acarbose solution or compounds 1-27 were added, then 30 µL of 0.6 U/mL α-glucosidase was pipetted and mixed by light shake. The 96-well plates were incubated in a constant-temperature incubator for 20 min at 37 • C, then 20 µL of 2.658 mmol/L p-PNG was added, and then incubation continued for another 28 min. The termination of the reaction was conducted by adding 80 µL of 0.2 mol/L Na 2 CO 3 to each well. The absorbance was recorded for each well at 405 nm by a microplate reader, and the experiment was carried out three times in parallel. The measured absorbance values were processed by Graphpad Prism software 9.5 to calculate the IC 50 values of the compounds.

Conclusions
In this study, two new compounds, named cajanstilbenoid C (1) and cajanstilbenoid D (2), together with eight other known stilbenoids (3-10) and seventeen known flavonoids (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27), were isolated. α-Glucosidase inhibition assay of all the compounds showed that 3, 8-9, 11, 13, 19-21, and 24-26 demonstrated robust inhibitory activity. By comparing their IC 50 values, the inhibitory effects of flavonoids against α-glucosidase were stronger than those of the stilbenoids, which might be due to their different skeletons. For stilbenoids, the activity of the ones that have hydroxy and methoxy substituents at C-3 and C-5 is relatively high, and the activity of the one possessing a methoxy group at C-5 is stronger than the one with a hydroxyl group at this position. In addition, the isopentenyl substituent favors activity, and when ring A contains a carboxyl or ester group, the activity decreases. For flavonoids, their type (flavones or isoflavones), glycosylation, and substituents (positions of hydroxy, methoxy groups, and isopentenyl moiety) all affect their inhibitory effects against α-glucosidase. Basically, the activity of flavones was stronger than that of isoflavones, and when the hydroxyl groups were substituted at C-5 and C-7, and the isopentenyl group was substituted at C-8, the inhibition effect was enhanced, whereas the activity was reduced when the hydroxyl group was glycosylated. The activity of the B ring with the hydroxyl group on C-3 and C-4 was stronger than that of no substituent on the B ring.
This study enriched the number of compounds isolated from the leaves of Cajanus cajan (L.) Millsp. and revealed some promising α-glucosidase inhibitors, which could be helpful for better utilization of this plant; yet, more research is needed to demonstrate if the α-glucosidase inhibitors obtained herein could work in cell or animal models to treat diabetes.